1. Field of the Invention
This invention relates to an improved support for use in solid-phase immunoassays for determining the presence and concentration of protein antigens in a liquid sample.
2. Description of the Prior Art
In recent years, numerous immunoassay techniques have been developed to simplify operating procedures of existing methods and to provide new methods of improved speed, sensitivity and accuracy. In particular, solid-phase reactions have been especially valuable in making possible procedures that could not be performed with conventional homogenous phase reactions.
A solid-phase reaction is generally carried out between one reactant, the fixed component, immobilized on the surface of an insoluble support matrix, and a second reactant, the mobile component, in solution. Typically the reactants are immunological counterparts, which may be alternately disposed as either the fixed or the mobile component. As used herein, the term "immunological counterpart" denotes either an antigen or an antibody which reacts specifically with the corresponding antibody or antigen.
The reaction may be a conventional chemical reaction, a binding of the mobile component by the fixed component as in an immunochemical reaction between an antigen and an antibody, or it may be a binding of the mobile component by the fixed component accompanied by chemical transformation of one of the components such as occurs in an enzyme-catalyzed reaction. Quantitative results are obtained by measuring the formation of products or disappearance of reactants as in the case of conventional and enzyme-catalyzed reactions, and in measuring the amount of the mobile component bound or the amount of mobile component unbound, in the case of an immunochemical reaction.
Where the reaction consists of binding, in the absence of chemical change, techniques developed in the field of immunochemistry may be used to measure the extent of the reaction. Solid-phase reactions are especially suited for immunochemical assays because the reactants in bound form may readily be removed from the solution by virtue of their attachment to the solid phase. A variety of solid-phase supports have been developed and used in the prior art.
In solid-phase technology, the reagent or reagents used in the procedure are usually immobilized by being coated or bonded, either covalently or by adsorption to the solid-phase material, which is then immersed in the sample to be tested.
Examples of commonly used solid-phase materials include, but are not limited to, glass or polymeric tubes which are coated with the reagent or reagents on their internal surfaces; coated polymeric inserts; coated polymeric sticks; micro and macro beads formed by polymers and of glass; porous matrices; coated membranes; and tablets.
The earliest solid-phase systems were test tubes coated on the inside surface. Various improvements on these coated tubes resulted in tubes having a convoluted surface to increase surface area. Additionally, tubes were provided with detachable lower sections which may be both coated to achieve greater uniformity from tube to tube. This batch immobilization results in the outsides as well as the insides of the tubes being coated, and thereby wastes valuable immunological reagents.
The following U.S. Patents are noted to be representative of the prior art: U.S. Pat. No. 4,378,344, to Zahradnik et al.; U.S. Pat. No. 4,147,752, to Souvaniemie et al; U.S. Pat. No. 4,210,418, to Brown et al; U.S. Pat. No. 4,180,383, to Johnson; U.S. Pat. No. 4,170,454 to Meriadec et al; U.S. Pat. No. 4,280,816, to Elahi; U.S. Pat. No. 4,225,784 to Barrett; U.S. Pat. No. 3,826,619 to Bratu, Jr. et al; U.S. Pat. No. 3,793,445 to Updike et al.; U.S. Pat. No. 4,066,512 to Lai; and, U.S. Pat. No. 3,951,748 to Devlin.
Currently, solid-phase assays utilize the physical absorption of the substance to be assayed (typically, an antibody) out of solution onto the inside surface of plastic test tubes. Frequently, the surface is treated or pretreated with a bifunctional coupling agent (such as glutaraldehyde) to improve the capacity and retention of the substance being assayed.
This type of immunoassay is subject to a number of disadvantages. For instance, only a certain type of plastic test tube will work and there is a binding variation since the plastic composition and molding characteristics of the test tube affect the binding ability of the substance being assayed. In the case where antibodies are the substance being assayed, there are the additional problems that physical absorption sometimes destroys the function of a percentage of antibody and/or may cause antibody leaching.
In addition, the tubes may not be stable for an extended period of storage, and are, in fact, frequently post-coated with a carrier protein such as bovine serum albumin or soluble gelatin for a hydrophilic protecting layer to enhance shelf-life. Generally, such tubes have a finite binding capacity (usually only about 1 .mu.g of antibody can be absorbed to 1 cm.sup.2 the inside surface of a plastic test tube) which limits the use of this technology to high titer antisera. Finally, the manufacture of such tubes in a consistent process requires costly equipment and much labor.
Various types of solid-phase matrices designed to be inserted into the reaction fluid have been disclosed in the prior art. These include sponge matrices, microporous membranes, closely-fitting inserts which squeeze the reactions fluid into a thin layer, disc shaped inserts, micro glass beads, coated macro beads and inserts having a plurality of water-insoluble fins. Many of these devices are illustrated in the U.S. Patents listed above, and to such extent, the disclosures of such patents are incorporated herein by reference.
All of the types of coated tubes and inserts suffer from two major disadvantages--excessive cost of manufacture and inconsistency between batches due to the multiple steps of manufacture. A need therefore exists for the development of a solid-phase support that may be uniformly, inexpensively and reliably manufactured and used.